PO:30:152 | An altered immunometabolic profile characterizes B cells of patients with oligoarticular- and polyarticular- juvenile idiopathic arthritis

Emiliano Marasco¹, Angela Aquilani¹, Rebecca Nicolai¹, Maria Isabella Petrone¹, Giusyda Tarantino¹, Ivan Caiello¹, Sabina Barresi², Silvia Magni Manzoni¹, Fabrizio De Benedetti¹ | ¹Unità di Reumatologia, Ospedale Pediatrico Bambino Gesù Roma, Italy; ²UO di Anatomia Patologica, Ospedale Pediatrico Bambino Gesù Roma, Italy

Objectives. An hyperactivation of B cells characterizes patients with oligoarticular- and polyarticular-(o-/p-)JIA (Barnes MG,A&R,2010). We aimed to investigate the signalosome and transcriptomic profile of B cells in patients with o-/p-JIA.

Methods. We enrolled patients with a diagnosis of o-/p-JIA (n=15) and age matched controls (n=15). Intracellular levels of phospho-proteins (Syk, BLNK, ERK1-2, BTK, PLC) and surface levels of CD22 were evaluated by flow cytometry. Calcium mobilization after anti-Ig stimulation was evaluated with Indo1 by flow cytometry. Transcriptomic analysis was performed on sorted naive B cells of patients and controls activated with anti-Ig, rhCD40L and IL-21. Total RNA was extracted with Trizol. Libraries were prepared with SureSelect-XT-HS2-RNA-System (Agilent), obtaining a strand-specific library positively enriched for human exons. Samples were run on a NextSeq500/550(Illumina).

Results. We first analyzed the basal expression and phosphorylation levels of key proteins involved in the regulation of B-cell receptor (BCR) signaling (Syk, BLNK, ERK1/2) in B cell of o-/p-JIA patients and controls. We observed a higher frequency of phosphorylation of BLNK, but not of Syk and ERK1/2 in both naive and memory B cells compared to controls (Figure 1A). We observed a lower surface expression of CD22, a negative regulator of B cell signaling in all B cell subsets, in naive B cells and unswitched memory (UnswM), but not in switched memory B cells (SwM) (Figure 1B), suggesting that most alterations in BCR signaling are established at the mature naive stage. Upon activation with an anti-Ig, naive B cells showed higher phosphorylation of BTK (Figure 1C). Upon in vitro stimulation of BCR with anti-Ig, naive B cells of JIA patients showed a faster calcium response (Figure 1D). We focused on naive B cells and investigated the transcriptomic profile of activated naive B cells. After quality controls, gene annotation to the reference genome, counting and filtering for low expression genes, we identified 13161 genes. With a significance threshold of adjusted p-value set at 0.05, we identified 372 differentially regulated genes (DEGs) between patients and controls that seprated the two groups in a PCA analysis (Figure 1E). We performed on our set of DEGs over-representation analysis, showing that most of the DEGs were related to immune signaling of the TNF family, and genes involved in the metabolisms of amino acids and protein synthesis(Figure 1F).

Conclusions. Our data show that naive B cells of JIA patients exhibit an increased phosphorylation of BLNK together with a reduced expression of CD22 and respond more promptly to BCR activation with faster calcium mobilization. Transcriptomic analysis of activated naive B cells revealed differential expression of genes associated with TNF family immune signaling, NF-kB pathways, antigen presentation, and amino acid metabolism, as well as those involved in protein synthesis.